Normal bone remodeling is a process in which new bone deposition by osteoblasts is balanced through bone resorption by osteoclasts (see Gowen, M., Every, J G, and Kumar S. Emerging therapies for osteoporosis. Emerging Drugs, 2000 5(1): p. 1-43.) In several disease states, the balance between bone deposition and bone resorption is perturbed. In osteoporosis, for example, excess bone resorption leads to brittle bones and frequent fractures of the wrist, vertebrae, and hip. In rheumatoid arthritis, increased bone resorption leads to malformations of the bones within arthritic joints. Re-establishing normal bone remodeling in these and other disorders can be achieved by decreasing or increasing the number and activity of osteoclasts (See Rodan, G A, and Martin T J. Therapeutic approaches to bone disease. Science 2000 289: p. 1508-1514.)
Several proteins modulate the bone remodeling orchestrated by osteoblasts and osteoclasts. (See US 2003/0013651 and WO 02/080955). Three key proteins are the cell-surface receptor RANK (Receptor Activator of NF-κB), the soluble decoy receptor OPG (osteoprotegerin), and the soluble and transmembrane forms of RANKL (RANK ligand, also known as RANKL, TNF-related activation induced cytokine (TRANCE), osteoclast differentiation factor (ODF), and osteoprotegerin ligand (OPGL). RANK is activated by the binding of its ligand, RANKL, which leads to the differentiation, survival, and fusion of pre-osteoclasts to form active bone resorbing osteoclasts (see Lacey D L, Timms E, Tan H-L, Kelley M J, Dunstan C R, Burgess T et al. Osteoprotegerin Ligand is a cytokine that regulates osteoclast differentiation and activation. 1998 Cell 93: p. 165-176.). RANKL is a trimeric TNF family member that binds to the trimeric RANK receptor.
The RANKL/OPG/RANK biochemical axis has been successfully targeted to treat osteoporosis, rheumatoid arthritis, prosthesis-induced osteolysis, cancer-induced bone destruction, metastasis, hypercalcemia, and pain (see Hofbauer, L C, Neubauer, A, and Heufelder A E. Receptor activator of nuclear factor-kB ligand and osteoprotegrin. 2001 Cancer 92(3): p. 460-470; Takahashi N, Udagawa N, and Suda T., A new member of the tumor necrosis factor ligand family, ODF/OPGL/TRANCE/RANKL, regulates osteoclast differentiation and function. 1999 Biochem. Biophys. Res. Comm. 256: p. 449-455). Therapies utilizing OPG (see Honore P, Luger N M, Samino M A C, Schwei M J et al. Osteoprotegerin blocks bone cancer-induced skeletal destruction, skeletal pain and pain-related neurochemical reorganization of the spinal cord. 2000 Nature Medicine 6(5):521-528.) or the soluble RANK-Fc protein (See Oyajobi B O, Anderson D M, Traianedes K, Williams P J, Yoneda T, Mundy G R. Therapeutic efficacy of a soluble receptor activator of nuclear factor kappaB-IgG Fc fusion protein in suppressing bone resorption and hypercalcemia in a model of humoral hypercalcemia of malignancy. 2001 Cancer Res 61(6): p. 2572-8; Childs, L M, Paschalis, E P, Xing L et al. In vivo RANK signaling blockade using the receptor activator of NF-kB:Fc effectively prevents and ameliorates wear debris-induced osteolysis via osteoclast depletion without inhibiting osteogenesis. J. of Bone and Mineral Res. 17: p. 192-199.) are also in development. OPG and soluble RANK-Fc protein constructs bind to RANKL, thereby decreasing amount of RANKL that is available for RANK receptor activation.
Hypercalcemia is a late stage complication of cancer, disrupting the body's ability to maintain normal levels of calcium, resulting in calcium deposit in the kidneys, heart conditions and neural dysfunction and occurs most frequently in patients cancers of the with lung and breast. Hypercalcemia also occurs in patients with multiple myeloma, cancers of the head and neck, sarcoma, cancers of unknown primary origin, lymphoma, leukemia, melanoma, renal cancer, and gastrointestinal cancers (e.g. esophageal, stomach, intestinal, colon and rectal cancers).
In addition to being important in bone biology, RANKL plays a role in the immune system by regulating antigen-specific T cell responses (See Anderson et al., A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function; Nature 1997, 390(6656):175-9). RANKL is highly expressed on activated T cells while the RANK receptor is expressed at high levels on mature dendritic cells (DC). The interaction between RANKL and RANK acts as a co-stimulatory signal, which enhances DC survival and T cell proliferation by inducing DC differentiation, cytokine production and reduced apoptosis in both cell types. Immunotherapy to produce tolerance to transplanted tissues and/or organs can be achieved by blocking the co-stimulatory signal using RANK antagonists. Blocking co-stimulation prevents T cell activation by DCs, and causes alloreactive T cells to become anergic and/or undergo apoptosis (See Adler et al., Immunotherapy as a means to induce transplantation tolerance; Current Opinion in Immunology 2002, 14:660-665). By a similar mechanism of action, antagonizing RANK signaling could be a treatment for autoimmune disorders such as systemic lupus erythematosus, inflammatory bowel disease, diabetes, multiple sclerosis, rheumatoid arthritis, and ankylosing spondylitis. (See WO 03/033664) RANKL variant superagonists, on the other hand could be used to activate the immune system by promoting T cell activation. RANKL superagonists could be useful treatments for diseases including but not limited to cancer and viral infection.
Much work has been done to develop therapeutic entities and reagents for biological research based on RANKL. For example, RANKL fragments, analogs, derivatives, or conformers having the ability to bind OPG, which could be used as treatments for a variety of bone diseases, have been described (See U.S. Pat. No. 5,843,678). RANKL variants, which induce production of an immune response that down-regulates RANKL activity, have been disclosed (See WO00/15807). In other studies, utilization of RANKL protein and its derivatives as immune modulators has been proposed (See WO99/29865). All references cited herein are hereby expressly incorporated by reference.
OPG is a natural antagonist of RANKL and functions as a negative regulator of osteoclastogenesis. OPG functions by binding and sequestering RANKL thereby preventing binding to and activation of RANK.
Accordingly, a need exists for RANKL antagonists and superagonists that do not bind OPG. These variants will have improved therapeutic efficacy, as they substantially reduce or eliminate sequestration by the decoy receptor, OPG and they substantially reduce interference with endogenous OPG from negatively regulating osteoclastogenesis